Fig. 4

Effect of doxorubicin treatment in Oci-Ly-19 after entinostat treatment on DNA repair mechanisms quantified by immunofluorescence assay. a Schematic representation of the experimental procedure prior immunofluorescence staining. Cell lines were treated with epigenetic inhibitors (i.e., entinostat, belinostat, vorinostat, or tazemetostat) at days 0, 3, 6, and 9, while a copy was kept untreated as control. After 9 days, both treated and control cell lines either received no doxorubicin or were exposed to doxorubicin for 4 and 24 h. Cells were then stained using immunofluorescent antibodies and imaged. Cells not receiving any treatment served as negative control, while cells not pretreated with epigenetic inhibitors but treated with doxorubicin served as positive control. b Immunofluorescence images of doxorubicin treated cells after entinostat treatment (above) and untreated (below). Composite image includes also DAPI (blue). c Quantification of proportion of cells positive for each marker in each image. The markers shown quantify apoptosis (cCasp3), DNA damage (gH2Ax), and homologous recombination (RAD51) (see Methods). Entinostat-induced sensitization to exogenous DNA damage is detected as reduced RAD51 foci, increased cCasp3 positivity and increased gamma-H2AX positivity in doxorubicin-treated cells. Asterisks indicate significant regulation (p < 0.05). All measurements from the immunofluorescence assay are shown in Additional file 1: Figure S4